Cytochrome P450 2E1 variable number tandem repeat polymorphisms and health risks: A genotype-phenotype study in cancers associated with drinking and/or smoking

Cytochrome P450 2E1 (CYP2E1) is one of the main enzymes involved in the oxidation of ethanol and in the transformation of a number of potentially dangerous compounds. It has various polymorphic sites, one of which is a variable number tandem repeat (VNTR) polymorphism previously described in the 5'-flanking region. The aim of this study was to investigate the genotype-phenotype association between CYP2E1 VNTR polymorphisms and risky health habits in healthy subjects and to analyze the associations between these polymorphisms with drinking- and/or smoking-related cancers. We analyzed 166 healthy subjects by genotyping for the CYP2E1 VNTR polymorphism associated with drinking and/or smoking habits by the more sensitive restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method, using the NlaIV restriction enzyme. Sixty cases of pancreatic adenocarcinoma (PA) and 66 with hepatocellular carcinoma (HCC), were also genotyped. Statistical analysis was carried out to investigate the genotype-phenotype associations and to compare certain genotypes and cancer. We found 7 genotypes both in the healthy subjects and patients. The A1/A1 genotype was observed to be mainly associated with non-drinkers and -smokers (87.5 and 75.0%, respectively); moreover it was never found in the PA or HCC patients. Conversely, a weak association between A2/A3 with smokers (45.8%) and A4/A4 with drinkers (53.9%) was detected. In addition, the A4/A4 genotype was found to be significantly associated to PA [odds ratio (OR)=3.25; 95% confidence interval (CI) 1.21-7.50]. Our data demonstrate that certain CYP2E1 VNTR genotypes are associated with drinking and/or smoking habits; consequently, they may contribute either to the decreased or increased risk of developing drinking- and/or smoking-related cancers. In particular, we hypothesize that the A1/A1 VNTR genotype may have a protective role against drinking- and/or smoking-related cancers, and that A4/A4 may be a high-risk genotype during the early stages of cancer

ALLELIC VARIANTS OF CYP2E1 GENE IN HEPATOCARCINOMA PATIENTS AND IN HEPATIC TUMOR CELL LINES

Background and Aims: Hepatic enzyme CYP2E1 is involved in the metabolism of a number of exogenous and endogenous substances (i.e. ethanol, drugs and chemical carcinogens). Being polymorphic, CYP2E1 gene can give different xeno-metabolic capabilities in a population and it is well known that inadequate or no enzymatic deactivation of xenobiotics could induce an increased susceptibility to disease and cancer. In particular, one of the 5 -flanking region polymorphisms, able to differentiate CYP2E1 gene transcriptional activity, is caused by the appearance/disappearance of RsaI and PstI restriction sites, which generates two different alleles, namely *C1(Rsa+/Pst−) and *C2(Rsa−/Pst+) respectively, reported to be in complete linkage disequilibrium. Methods: To confirm the existence of a correlation between some particular CYP2E1 genotypes/haplotypes and hepatocarcinoma, we determined CYP2E1 PstI/RsaI genotypes/haplotypes by RFLP-PCR in a cohort of central western Sicily hepatocarcinoma patients and in a population of healthy students from the same geographic area. Results: In hepatocarcinoma patients, modal genotype association was Rsa++/Pst−−, corresponding to CYP2E1 *C1/*C1 haplotype, whereas the Rsa+−/Pst−+ association, equivalent to CYP2E1 *C1/*C2 haplotype, resulted to have the lowest frequency both in patients and in controls. Moreover, both in patients and in controls, noncanonical genotype associations were frequent and arose from a no-linkage disequilibrium between the two polymorphic sites. Other authors reported this finding as a rare occurrence. Thus, from analysis of only one restriction site, Rsa++ genotype was approximately 1.5-fold more frequent in patients than in controls, and the non-canonical Rsa+− genotype was found relatively frequent in patients. Moreover, HuH7 and HA22T transformed hepatocarcinoma cell lines also showed the Rsa+− genotype. Conclusions: These results suggest that the presence in CYP2E1 genotype of at least one allele with an Rsa I restriction site is correlated with hepatocarcinoma. As this site is known a consensus sequence for some specific CYP gene transcription factors, like HNF-1, it may be supposed that a single nucleotide polymorphism can alter the possibility of HNF-1 to bind CYP2E1 promoter. This could determine a marked change in the transcriptional activity of the gene, incompetence in xenobiotic metabolism or in toxic substance deactivation and an increased susceptibility to neoplastic diseases, such as hepatocarcinoma

"Global change, sostenibilità ambientale e Biodiversità": il PCTO delle classi III D e III E, indirizzo (Scienze Applicate) del Liceo Scientifico "Pietro Ruggeri" di Marsala presso l'IAS - CNR Sede Secondaria di Capo Granitola

Con la partecipazione ad Esperienza InSegna 2020 (https://www.esperienzainsegna.it/) - Cambiamento climatico e sostenibilità ambientale - organizzata dall'Associazione PALERMOSCIENZA, da martedì 18 a domenica 23 febbraio 2020 presso l'Università degli Studi di Palermo (Edificio 19 - Viale delle Scienze) - l'IAS – CNR porta a termine un'esperienza formativa di successo rivolta a 54 allievi delle classi III D e III E, indirizzo Scienze Applicate, del Liceo Scientifico "Pietro Ruggeri" di Marsala (TP) presso l'IAS – CNR – S. S. di Capo Granitola nell'ambito del Percorso per le competenze trasversali e per l'orientamento (PCTO ex ASL), e regolamentata dalla convenzione Prot. IAS n° 0000030 del 08/01/2020. Il Direttore f.f. di IAS – CNR il Dottor Mario Sprovieri e il Responsabile dell'IAS – CNR Sede Secondaria di Capo Granitola, il Dottor Giorgio Tranchida, hanno promosso e supportato le attività del PCTO, anche quest'anno come negli anni passati, accogliendole come una irrinunciabile occasione per i tecnici, i tecnologi e i ricercatori di perseguire la " terza missione" degli Enti di Ricerca, attraverso l'applicazione diretta, la valorizzazione e l'impiego della conoscenza per contribuire allo sviluppo sociale, culturale ed economico della società. Sono state 25 le unità di personale (8 ricercatori, 2 tecnologi, 11 tecnici e 4 assegnisti di ricerca) dell'IAS – CNR, Sedi Secondarie di Capo Granitola e di Palermo, che hanno lavorato in sinergia e con entusiasmo, presso la S. S. di Capo Granitola, permettendo di elaborare una proposta formativa ricca di contenuti scientifici da trasferire agli alunni delle classi III D e III E del Liceo Scientifico "P. Ruggeri". Il personale scientifico, grazie all'esperienza acquisita negli anni, è ben consapevole del fatto che le pratiche attive, come l'apprendimento collaborativo ed i piccoli gruppi di lavoro, risultano altamente produttive permettendo all'alunno di non acquisire solo conoscenze, ma soprattutto abilità e competenze. Al fine di accrescere le opportunità di conoscenza e accendere l'interesse negli studenti, il PCTO, dal titolo "Global change, sostenibilità ambientale e Biodiversità", è stato programmato e realizzato attraverso una prima parte seminariale, un'esperienza teorico-pratica su campo (per prelevare campioni da analizzare) e una parte laboratoriale. Successivamente: i) una parte relativa a seminari teorico-pratici riguardante l'analisi statistica dei dati scientifici; ii) un gioco di ruolo finalizzato all'apprendimento collaborativo relativo dei concetti scientifici acquisiti ed, infine iii) una visita presso l'Osservatorio Regionale della Biodiversità Siciliano (ORBS) che è ospitato presso la struttura di Capo Granitola. Il personale scientifico ha rivestito un ruolo importante, delicato, di grossa responsabilità nell'accompagnare gli alunni in questo percorso di orientamento (PCTO) e affinché tutti gli alunni "imparassero ad imparare" nel modo per loro più corretto, hanno concordato nell'utilizzare diverse metodologie didattiche efficaci quali: interdisciplinarietà, utilizzo di parole chiavi, cooperative learning, didattica laboratoriale, peer education, problem solving, role playing, studio di caso, discussione, project work. Le metodologie didattiche utilizzate hanno consentito di valorizzare il potenziale di apprendimento

Association between single nucleotide polymorphisms in the COX-2, TNF-a and VEGF-A genes, and susceptibility to hepatocellular carcinoma

Introduction: TNF-a, COX-2 and Vascular Endothelial Growth Factor (VEGFA) are mediators of inflammation and angiogenesis, all of them are abundantly produced in liver cirrhosis (LC). It was proposed that there is an association between single nucleotide polymorphisms (SNPs) and hepatocellular carcinoma (HCC). These allelic variants influence the transcriptional activity of these genes, and therefore the proteins levels. The VEGF-A pathway is a potential therapeutic target in HCC, and several anti-angiogenic agents have entered clinical trials in HCC. Aims: 1) To evaluate thè frequency of SNPs of COX-2, TNF-a and VEGF-A genes in patients with HCC vs LC patients and a control group (C). 2) To verify thè correlation between thè allelic variations and the risk of developing HCC tumors. Methods: DNA extracted from whole blood of C, HCC and LC patients (all from Sicily, Italy), was used for the evaluation of SNPs by the RFLP-PCR method. The SNPs analyzed were at position -1195 G>A in the promoter regions, of COX-2 (A allele is associated with higher levels of thè enzyme), at position -308 G>A in thè promoter regions of TNF-a genes (G allele is associated with lower levels of the cytokine), and at position +936 C>T in the coding region of VEGFA gene (T allele is associated with high levels of the growth factor). X2, Fisher exact tests were used for statistical analysis. Results: We obtained a significant difference only for the SNP +936 C>7 of VEGF-A gene. Results obtained are shown in the following table

CYP2E1 VNTR polymorphisms and hepatocarcinoma: a gender-specific correlation

Cytochrome P450 (CYP2E1) is often associate to susceptibility to alcohol-related diseases and various cancers, because of its role in the metabolism of multiple environmental xenobiotics. In the 5’- flanking region of the human CYP2E1 gene there are restriction fragment length polymorphism which are involved in the transcriptional regulation of the CYP2E1 gene. Recently a tandem repeat polymorphism (VNTR) in the 5’-flanking region of CYP2E1 was found. Because cytochrome P450 2E1 catalyzes the metabolic activation of pro-carcinogen and cytotoxic compound, we value the genetic distribution of this tandem repeat polymorphism in a healthy population, and in patients with hepatocellular carcinoma living in same country, in order to found a correlation between CYP2E1 VNTR genotype and neoplasia. DNA was isolated from spit sample of 108 control subject and from the peripheral lymphocytes of 35 HCC patients. The 5’ flanking region of the CYP2E1 gene was amplified by polymerase chain reaction and examined for tandem repeat polymorphism using Nla IV restriction map. This study reports that only four of the ten possible genotype were found in all subjects. The modal genotype, found in both analyzed populations, is A2/A2. Interestingly, in a gender-based analysis of data this genotype was found more frequent in woman with disease that in control ones. These preliminary findings represent a first report of a gender-specific correlation between CYP2E1 VNTR polymorphism and hepatocarcinoma

Association Between Single Nucleotide Polymorphisms in the Cyclooxygenase-2, Tumor Necrosis Factor-a, and Vascular Endothelial Growth Factor-A Genes, and Susceptibility to Hepatocellular Carcinoma

Cyclooxygenase-2 (COX-2), vascular endothelial growth factor-A (VEGF-A), and tumor necrosis factor-a (TNF-a) are mediators of inflammation and angiogenesis; all of them are produced in liver cirrhosis (LC) and in hepatocellular carcinoma (HCC). It was proposed that there is an association between single nucleotide polymorphisms (SNPs) and HCC. These allelic variants influence the transcriptional activity of these genes, and therefore the proteins levels. The VEGF-A pathway is a potential therapeutic target in HCC, and several antiangiogenic agents have entered clinical trials in HCC. We evaluated the frequency of SNPs of COX-2, TNF-a, and VEGF-A genes in patients with HCC versus LC patients and a control group. The aim of this article was to verify the correlation between the allelic variations and the risk of developing HCC. The study included 96 HCC, 79 LC patients, and 162 healthy subjects. The evaluation of SNPs was performed by the restriction fragment length polymorphism (RFLPPCR) method. The SNPs analyzed were: 1195G>A of the COX-2 gene, 308G>A of the TNF-a gene, and +936C>T of the VEGF-A gene. Chi-square and Fisher exact tests were used for statistical analysis. Our results confirm that carriers with the C allele in the VEGF-A gene are more frequent in HCC versus LC ( p¼0.039), suggesting that this SNP may predispose to the development of HCC